This application addresses broad Challenge Area, (08) Genomics and Specific Challenge Topic, 08-ES-106: The role of environmental exposure in copy number variation (CNV): "Microscopic deletions and replications of the genome have attracted increasing attention for their potential role in many complex human diseases. Of particular interest are spontaneous CNVs, defined as those present in an affected individual, but absent in both parents. There is limited understanding of how spontaneous CNVs arise. Studies are needed that will determine whether environmental exposures can affect risk for copy number variation and other structural variations that have been implicated in complex diseases. Given the early stage of this research area, studies should focus on changes in cells exposed in vitro". In recent years, copy number variants (CNVs) have been found to be widely distributed throughout the human genome where they represent an important component of genetic variation and play an integral role in phenotypic diversity, complex disease and evolution. Over 1300 CNVs with frequencies of >1% have been described in healthy individuals that exist as deletions or duplications ranging in size from a few Kb to over a Mb, and this number will surely increase as more data become available. Similar spontaneous CNVs are now well known to be a major cause of genetic and developmental disorders, including mental retardation, autism, schizophrenia, epilepsy, cleft palate and many others. Studies of idiopathic mental retardation and developmental disorders have found de novo CNVs in 5-17% of affected individuals, suggesting a high mutation rate. Similar copy number alterations are also found at high frequency in many cancers where a role in initiation or progression is likely. CNVs are thus a key factor in normal genetic variation and evolution and are a very important class of mutation in genetic and developmental disorders and cancer. There is very limited understanding of how CNVs arise, the cellular mechanisms and risk factors that are involved, and the effects of environmental agents on their formation. As with all mutation classes, it is almost certain that environmental insults can induce or increase the risk for new and deleterious CNVs, however we have little knowledge of the mechanisms and frequency of such events. Meiotic unequal recombination [or non-allelic homologous recombination (NAHR)] mediated by flanking repeated sequences or segmental duplications leads to many recurrent, disease-related CNVs. However, there is growing evidence that many or most normal and sporadic, nonrecurrent CNVs, which account for the majority of disease-associated CNVs in humans and those in cancers arise via mechanisms coupled to aberrant DNA replication and/or non-homologous repair of DNA damage. This suggests an unexpected mitotic, rather than meiotic, cell origin for many CNVs and has a number of important implications for the role of environmental exposures in their formation and the development of in vitro model systems for their study. The Challenge, and our goal, is to determine the role of environmental factors in the formation of CNVs and to gain novel insight into the mechanisms by which these frequent mutations are generated. We have assembled a strong team of investigators and have developed a normal human cell culture model system coupled with leading edge genome analysis technologies, placing us in a unique position to address this timely Challenge. Using this system we have found that aphidicolin-induced replication stress leads to a remarkably high frequency of de novo CNVs. These findings lead us to hypothesize that environmentally-induced replication stress and/or DNA double strand breaks are two major factors leading to CNVs during mitotic cell divisions in the human germline and in cancer cells. To test this hypothesis, we will characterize environmental agent-induced CNVs at the genome-wide scale and directly compare the strength of these two non-exclusive models for CNV formation, thus providing for the first time a high resolution catalog of genomic manifestations of two different categories of environmental agents most likely to be associated with CNV formation. These are (1) agents that lead to replication stress, which might lead to CNVs through secondary breakage or replicative template switching, and (2) agents that directly induce DNA double-strand breaks (DNA DSBs), which might lead to CNVs through inappropriate joining of broken ends. Specifically, we will examine hydroxyurea and folate stress, which inhibit replication through different mechanisms than aphidicolin, and on ionizing radiation and bleomycin, which lead directly to DNA DSBs. Our "shovel ready" in vitro model system coupled with high resolution genomic microarrays and next-generation sequencing will allow us to determine the effects of these agents on the frequency, spectrum, distribution and structure of CNVs and other submicroscopic structural variations. The combined results will address a major gap in our knowledge about a very important class of mutations and allow predictions of the environmental agents that confer the greatest risk for ongoing structural alteration of the human genome.